The emerging relationships between epigenetics, gametogenesis, and cancer provide the impetus for the utilization of chromatin remodeling agents for the treatment and prevention of lung cancer.Previously, we reported that the DNA demethylating agent, 5 aza-2'deoxycytidine (DAC) and the HDAC inhibitor, Depsipeptide FK228 (DP) synergistically induce apoptosis, and markedly enhance NY-ESO-1 expression preferentially in cancer cells, facilitating their recognition by cytolytic T lymphocytes (CTL) specific for this CTA. Furthermore, we have observed that adoptive transfer of CTL recognizing a CTA induced by DAC in vivo diminishes pulmonary metastases in a syngeneic murine tumor model. These preclinical data provided the rationale for several protocols have been conducted in the Thoracic Oncology Section, Surgery Branch, NCI in an attempt to recapitulate in clinical settings drug exposure conditions that mediate apoptosis and CTA induction in cultured lung cancer cells. To date, more than 80 patients have received DAC, DP, or sequential DAC/DP infusions on these protocols. Clinical toxicities, and responses to therapy have been assessed by CTCAE and RECIST criteria, respectively. Plasma DAC and DP levels have been evaluated by LC-MS and HPLC techniques. Quantitative RT-PCR, methylation-specific-PCR, immunohisto-chemistry, and ELISA techniques have been used to assess a variety of molecular endpoints in pre-and post-treatment tumor biopsies and sera. Micro-array techniques have been used to comprehensively examine gene expression profiles in laser-captured tumor cells from pre- and post treatment biopsies from 21 individuals receiving DAC, DP, or sequential DAC/DP infusions. Results of these arrays have been compared to data derived from analysis of laser-captured tumor cells and adjacent, histologically normal bronchial epithelia from 20 patients undergoing definitive lung cancer resections. Whereas no objective responses have been observed, several patients have exhibited prolonged stabilization of disease following DAC, DP, or sequential DAC/DP infusions. Plasma DAC and DP concentrations have approximated threshold levels for gene induction and apoptosis in cultured lung cancer cells. Approximately 30% of patients receiving DAC or DP infusions have exhibited enhanced expression of NY-ESO-1, p16, p21, or acetylated histone H3 in tumor biopsies. Long-oligo array analyses have revealed complex, heterogeneous responses to DAC, DP, and DAC/DP in lung cancer cells, with an apparent shift of gene expression profiles toward those observed in histologically normal bronchial epithelia. Collectively, these data confirm that DNA demethylating agents and HDAC inhibitors can modulate gene expression in primary lung carcinomas, and support evaluation of these compounds in conjunction with other treatment regimens targeting induced gene products or survival pathways in lung cancer cells.Because CTAs are normally expressed only in germ cells, evaluation of the mechanisms that facilitate de-repression of C-T genes may provide considerable insight regarding the DNA methylation paradox in cancer cells. As such, additional laboratory studies have focused on the mechanisms regulating NY-ESO-1 expression in lung cancer cells and normal respiratory epithelia. Recently we have shown that the unique pair of paralogous transcription factors, CTCF and BORIS, which previously have been implicated in epigenetic regulation of imprinting and X chromosome inactivation, directly modulate NY-ESO-1 (as well as MAGE-A1) expression in lung cancer cells. Our experiments indicated that NY-ESO-1 expression coincided with de-repression of BORIS in cultured lung cancer cells. Quantitative RT-PCR analysis revealed robust, co-incident induction of BORIS and NY-ESO-1 expression in lung cancer cells, but not NHBE cells, following DAC, DP, or sequential DAC/DP exposure under clinically relevant conditions